The goal of many analytical chemistry protocols is to separate a sample (blood, tears, urine, water from a well, etc.) into its individual components or constituents so that each component may be evaluated without any interference from other components. A variety of techniques have been developed for this task and include electrophoretic protocols, chromatographic protocols, and the like. Once the constituents have been separated, they can be detected by various techniques, e.g., refractive index, electrochemical, or ultraviolet-absorbance, which can indicate the presence of a given constituent. The amount of constituent may be determined by the intensity of the signal produced in a detector. A detector is employed to measure a signal peak as each constituent exits the column and may be “on-line” or “off-line” (i.e., integral with the separation apparatus or a separate component thereof, respectively). By comparing the time it takes for the peak to show up (also referred to as the retention time) with the retention times for a mixture of known compounds, the constituents of unknown sample mixtures can be identified. By measuring the signal intensity (also referred to as the response) and comparing it to the response of a known amount of that particular analyte, the amount of analyte in the mixture can be determined.
For example, one technique that is often employed to separate various constituents of a sample from each other is chromatography, where liquid chromatography (“LC”) is often employed. Liquid chromatography is an analytical chromatographic technique that is useful for separating ions or molecules that are dissolved in a liquid or solvent. If the sample solution is in contact with a second solid or liquid phase, the different solutes will interact with the other phase to differing degrees due to differences in adsorption, ion-exchange, partitioning, or size. These differences allow the mixture components to be separated from each other by using these differences to determine the transit time of the solutes through a column. Chromatography may be coupled with a suitable on- or off-line detection system that can characterize each type of separated constituent. One liquid chromatography protocol that is often employed due to its versatility is high performance liquid chromatography (“HPLC”). Generally, HPLC includes passing a sample of constituents in a high pressure fluid or solvent (called the mobile phase) through a tube or column. The column is packed with a stationary phase. The stationary phase is typically composed of a substrate or matrix such as particles, e.g., porous beads or the like. The pore sizes can be varied to allow certain sized analytes to pass through at different rates. As the constituents pass through the column they interact with the mobile and stationary phases at different rates. The difference in rates is due to the difference in one or more physical properties of the constituents, e.g., different polarities. The constituents that have the least amount of interaction with the stationary phase, or the most amount of interaction with the mobile phase, will thus exit the column faster.
One particularly useful mode of HPLC—particularly for the separation of highly polar or ionizable constituents, is reversed phase high performance liquid chromatography (“RP-HPLC”). RP-HPLC primarily operates on the basis of hydrophilicity and lipophilicity to separate various constituents of a liquid medium from each other. The stationary phase includes a substrate (which may be a plurality of particles) that has bound chemical moieties (i.e., a bonded phase), such as hydrophobic chains, e.g., bound alkyl chains, and the like, which facilitate the separation of the constituents. Accordingly, the greater the hydrophobicity of the bound chemical moieties, the greater is the tendency of the hydrophobic constituents in the mobile phase to be retained in the column while the hydrophilic constituents are eluted more rapidly from the column than the hydrophobic constituents.
Regardless of the analytical protocol or technique employed, oftentimes the sample of interest is associated with a fluid, i.e., a mobile phase, prior to the actual separation step, e.g., prior to being contacted with a matrix or solid phase such as a chromatographic column or channel or the like, e.g., a capillary channel such as employed in a microfluidic device. The mobile phase is often a particular ratio mix of two or more fluids and in many instances the mix ratio of the fluids of the mobile phase changes over the course of a particular protocol, e.g., to provide a gradient of the mobile phase over time. For example, in many protocols a mobile phase is composed of a mix of an organic solvent and an aqueous solvent, where in certain instances it may be desirable to vary the ratio of the two solvents over the course of the protocol to provide a gradient, e.g., gradually or step-wise. Accordingly, regardless of the type of analytical protocol employed, it is critical to the outcome of the protocol to know and maintain a particular mix ratio of the mobile phase at any given time point during a protocol.
However, it is difficult to determine the mix ratio of the mobile phase, e.g., prior to, during and/or after the separation step, to ensure the proper mix ratio is employed and/or to monitor the mix ratio, where such is particularly relevant in gradient elution wherein the mix ratio changes over time throughout a protocol and it is important to understand what the particular mix ratio is at a given time point of time. Although the mix ratio is known at the source (e.g., the pump that is used to deliver the fluid to the system), there is a delay between the setting of the mix ratio at the source and the time the ratio reaches the separation apparatus or a particular area of the separation apparatus such as the area where separation actually takes place, e.g., a separation column or channel. Accordingly, in order to know the exact conditions of a given separation protocol, the mix ratio must be measured at (i.e., on) the device itself- in other words in “real time”.
Accordingly, there continues to be an interest in the development of new methods and apparatuses for determining the mix ratio of a mobile phase. Of particular interest is the development of such methods and apparatuses that determine the mix ratio directly on a separation apparatus itself, are easy to use, and have a high degree of precision.
References of interest include: U.S. Pat. Nos. 4,399,036; 4,908,112; 5,770,029; 5,755,942; 5,746,901; 5,681,751; 5,658,413; 5,653,939; 5,653,859; 5,645,702; 5,605,662; 5,571,410; 5,543,838; 5,480,614; 6,046,056; 6,143,248; 6,158,712; 6,296,452; 6,375,901; 6,431,212; 6,495,016; 6,533,553; 6,561,224; and U.S. patent applicant Ser. Nos. 2003/0000835 and 2001/0048958. Also of interest are: Knox et al. (1987) Chromatographia 24:135); Peters et al. (1998) Anal. Chem. 70:2288); Hadd, et al., Microchip device for performing enzyme assays. Analytical Chemistry 69, 3407–3412 (1997); Macounova, et al. Concentration and separation of proteins in microfluidic channels on the basis of transverse IEF. Analytical Chemisty 73, 1627–1633 (2001); and Bucholz, et al. Microchannel DNA sequencing matrices with a thermally controlled “viscosity switch”. Analytical Chemisty 73, 157–164 (2001).